Flindissone, a Limonoid Isolated from Trichilia prieuriana, Is an LXR Agonist

In this study, the ability of six limonoids from Trichilia prieuriana (Meliaceae) to activate the liver X receptor (LXR) was assessed. One of these limonoids, flindissone, was shown to activate LXR by reporter-gene assays. Flindissone is a ring-intact limonoid, structurally similar to sterol-like LXR ligands. In endogenous cellular settings, flindissone showed an activity profile that is characteristic of LXR agonists. It induced cholesterol efflux in THP-1 macrophages by increasing the cholesterol transporter ABCA1 and ABCG1 gene expression. In HepG2 cells, flindissone induced the expression of IDOL, an LXR-target gene that is associated with the downregulation of the LDL receptor. However, unlike synthetic and similarly to sterol-based LXR agonists, flindissone did not induce the expression of the SREBP1c gene, a major transcription factor regulating de novo lipogenesis. Additionally, flindissone also appeared to be able to inhibit post-translational activation of SREBP1c. The results presented here reveal a natural product as a new LXR agonist and point to an additional property of T. prieuriana and other plant extracts containing flindissone.

Trichilia prieuriana A. Juss is an evergreen tree that grows in tropical Africa.It belongs to the genus Trichilia within the Meliaceae family.Leaves, stem, and root bark as well as root wood preparations have reported medicinal applications in treating various conditions, particularly bacterial and parasitic infections and pain or as a purgative. 1Members of the Meliaceae family characteristically produce limonoids, a group of highly oxygenated and modified triterpenes, as secondary metabolites. 2The prototypical limonoid structure (ring-intact skeleton) consists of a tetracyclic triterpene (A/B/C/D rings) and a 17β-furan ring, although the majority of the naturally occurring limonoids possess extensively rearranged skeletons. 2imonoids from the genus Trichilia were reported to have insecticidal, anti-inflammatory, and cytotoxic activities. 3Pagna et al. isolated and identified chemical constituents of T. prieuriana, which included the limonoids flindissone (1), deoxyflindissone (2), picraquassin E (3), and prieurianin (4).In their study, 1 showed strong antibacterial activity. 4he liver X receptor (LXR) is a member of the nuclear receptor family of ligand-activated transcription factors.It consists of three main domains: the N-terminal domain with a ligand-independent transactivation function, the DNA-binding domain (DBD), and the ligand-binding domain (LBD).To be transcriptionally active, the LXR has to form a heterodimer with the retinoid X receptor (RXR), which can also permissively activate the LXR heterodimer through its own ligand.There are two LXR isotypes, which differ mainly in their pattern of expression, with LXRα being the predominant form in the liver, intestine, and macrophages, while LXRβ is ubiquitously expressed. 5The endogenous ligands for LXR are most likely oxidized cholesterol derivatives, such as 22Rhydroxycholesterol (22R-OHC), 6,7 given that the LXR is a major regulator of cellular and whole-body cholesterol homeostasis. 8,9LXR regulates cholesterol absorption in the intestine, 10 reverse cholesterol transport from peripheral tissues and macrophages, 11,12 and cholesterol uptake by the liver. 13,14Conditions such as atherosclerosis are characterized by the accumulation of cholesterol-laden macrophages inside the vessel wall.Targeting LXR is considered a viable option to increase cholesterol efflux from atherosclerotic macrophages. 15,16However, synthetic LXR agonists, unlike endogenous oxysterols, induce hepatic steatosis and hypertriglyceridemia, due to activation of the LXR-SREBP1c pathway for de novo lipogenesis in the liver. 10,17,18In addition, the liver LDL receptor (LDLR) is subjected to negative regulation by LXR. 14,19Systemic LXR agonism can thus potentially lead to reduced LDL uptake by the liver and an increase in the blood levels of atherogenic LDL particles. 14,18,19imonoids isolated from T. prieuriana, especially those with a ring-intact structure (1−3, Chart 1), share a four-ring core structure similar to sterol-like LXR ligands.−33 This prompted us to investigate whether some limonoids are LXR agonists as well.

■ RESULTS AND DISCUSSION
The six limonoids 1−6 were assessed in luciferase assays for their ability to activate LXRα or LXRβ at concentrations between 1 and 10 μM.Only 1 was able to activate LXRs beyond the 2-fold threshold, while 3, 4, and 6 showed significant cytotoxicity (data not shown).Therefore, the subsequent characterization focused on 1 alone.Complete concentration−response curves of 1 in LXRα and LXRβ luciferase assays are shown in Figures 1A and 1B, respectively.
In comparison to the steroid LXR agonist 22R-OHC, 1 showed comparable potency and a somewhat smaller efficacy in activating the LXRα receptor, but it was much less effective in activating LXRβ.To verify that 1 binds to the LXR-LBDs, Gal4-luciferase assays were employed.The Gal4-luciferase assay requires the yeast response element UAS, which cannot be activated via cell-endogenous pathways.Additionally, a permissive contribution of RXR to the activation of the LXRresponse element can be excluded.Therefore, the activation of the hybrid receptor is an indication that the ligand binds directly to the LBD of the receptor.In comparison to the synthetic agonist GW3965, both steroid structures were much less effective in activating the Gal4 receptors but comparable to one another (Figures 1C and 1D).Compound 1 could activate both the LXRα-and LXRβ-Gal4 receptors in a concentrationdependent manner.However, a full sigmoidal curve could not be obtained due to the emergence of toxicity at concentrations higher than 10 μM (Figure S6, Supporting Information).

Journal of Natural Products
Finally, 1 did not activate other nuclear receptors: RXR, FXR, or RORγ (data not shown).
The X-ray crystal structure of the human LXRβ-LBD in complex with 24(S),25-epoxycholesterol (eCH), a potent endogenous oxysterol activator of LXR, reveals the binding mode of steroid LXR agonists to the ligand binding pocket (PDB 1P8D). 34Compound 1 likely binds to the LXR-LBD in a mode similar to eCH and 22R-OHC.−37 Both 1 and 22R-OHC produced stable poses that are similar to the experimentally observed binding mode of eCH (Figure 2).The Glide docking scores (i.e., estimated binding energies) were −9.65 and −10.80 kcal/mol for 1 and 22R-OHC, respectively, indicating good geometric matches of the predicted poses with the ligand binding site (the Glide docking score for redocked eCH is −10.74 kcal/mol).The hydroxyl moiety in position 3 of 22R-OHC is predicted to form hydrogen bonds with Asn239 and Phe329 via the H 2 O molecule HOH97.This is consistent with the hydrogen bonds observed for eCH in the X-ray structure.The presence of two methyl groups in 1 at position 4 hinders the formation of hydrogen bonds.Instead, the keto group in position 3 of 1 is likely to form hydrogen bonds with Ser278 and Glu281 via the H 2 O molecule HOH5 (Figure 2).The hydroxyl group in position 21 of 1 likely also plays a role in its bioactivity since 2, which lacks this group, was not active in luciferase assays.However, the role of this particular hydroxyl moiety in LXR binding could not be conclusively assessed with docking.It is also plausible that the hydroxyl group influences compound 1's biochemical properties such as intracellular transport and metabolism and, as a consequence, its availability for target binding.
Next, the ability of 1 to induce the gene expression of known LXR targets in an endogenous cellular environment was examined.LXR agonists are known regulators of the macrophage cholesterol efflux, where they upregulate gene expression of the cholesterol transporters ABCA1 and ABCG1. 11,12In the THP-1 macrophage cell line, 1 was able to stimulate plasma-mediated cholesterol efflux in a concentration-dependent manner (Figure 3A).At 5 μM concentration, 1 was more effective than 22R-OHC. 1 induced transcription of the ABCA1 gene (Figure 3B), which was accompanied by the corresponding increase in ABCA1 protein levels (Figure 3C).In addition, the ABCG1 gene was upregulated by 1 (Figure S7, Supporting Information).
In the liver, LXR activation regulates cholesterol uptake.LXR agonists have been shown to decrease protein levels of the LDL receptor and thus reduce LDL uptake. 14,19The mechanism behind it involves transcriptional upregulation of the inducible degrader of the LDLR (IDOL; official name MYLIP) gene, which is a direct LXR target. 14In the hepatoma cell line HepG2, 1 was not as potent or effective as GW3965, with only the highest concentration of 10 μM inducing a significant increase in IDOL expression (Figure 4A).None of the LXR agonists had any effect on the LDLR mRNA levels (Figure 4B).Unfortunately, it was not possible to detect the LDLR protein using Western blot in the HepG2 cell line due to the inconsistency of the employed antibodies (data not shown) and thus determine the effect of 1 on the LDLR protein levels.However, based on the published literature, 14,19 the LDLR protein levels are supposed to be decreased with GW3965 and 10 μM 1 treatments.Increased lipogenesis in the liver is a major side-effect of LXR activation. 10,17,18LXR agonists increase the expression of SREBP1c (SREBF1 gene), which is the master transcriptional regulator of fatty acid synthesis.For an active state, SREBP1c has to be cleaved from its 130 kDa precursor form located in the ER membrane to an active 60 kDa protein that can translocate into the nucleus. 38Unlike synthetic LXR agonists, sterol-like LXR ligands appear to be weaker activators of the SREBP1c transcription, particularly in liver cells, 24,26,27,29,32 although some ligands can also selectively upregulate SREBP1c expression 39 or function as context-specific antagonists. 40urthermore, sterols and oxysterols were shown to inhibit the proteolytic activation of SREBP1c by binding to the SREBP cleavage-activating protein (SCAP) and insulininduced gene 2 protein (INSIG2), respectively.In a sterolbound state, these two chaperones anchor the inactive SREBP1c precursor to the ER membrane. 41,42n HepG2 cells, only the synthetic agonist GW3965 induced an increase in cellular lipid content, as measured by the Oil Red O staining (Figure 5A).In comparison to GW3965, 1 significantly induced neither SREBF1 transcription (Figure 5B) nor increased protein levels of the 130 kDa precursor at any concentration (Figure 5C).However, 1 appeared to inhibit proteolytic processing of the SREBP1c, as shown by the concentration-dependent reduction of the 60 to 130 kDa protein ratio after 72 h of compound treatment, although the effects were not statistically significant (Figure 5D).In addition to SREBP1c, a similar proteolytic-activation mechanism also applies to SREBP2, a transcription factor involved in cholesterol synthesis. 41,42Since it was not possible to detect cleaved SREBP2 protein in HepG2 cells sufficiently to allow for quantification (data not shown), expression of its target gene 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) was measured instead.Compound 1 dosedependently also decreased HMGCR expression (Figure S8, Supporting Information).22R-OHC was reported to inhibit proteolytic processing of the SREBP2 protein at 20 μM; 42 however no inhibition of the proteolytic processing of SREBP1c nor a decrease in HMGCR gene expression could be observed here up to concentrations of 10 μM (data not shown).Since the nonsteroidal agonist GW3965 also reduced the HMCGR gene expression, the mechanism involved may not depend solely on the inhibition of SREBP2 proteolytic processing.
Taken together, these observations demonstrate that flindissone (1) is a new natural product, an LXR agonist.In vitro characterization of 1 showed an activity profile that corroborates its function as an LXR agonist.Like GW3965 and 22R-OHC, 1 induced ABCA1 and ABCG1 gene expression and cholesterol efflux from THP-1 M0 macrophages.In HepG2 cells, GW3965 and 1 induced IDOL expression, which is associated with the downregulation of LDL uptake in the liver.However, unlike GW3965, but similarly to 22R-OHC, 1 did not significantly induce SREBF1 expression and subsequent activation of de novo lipogenesis.In addition, 1 appears to be able to inhibit the proteolytic processing of SREBP transcription factors, most likely due to its structural similarity to sterols.However, 1 was not particularly potent in cellular assays, as the effects were most notable above 1 μM.This may be due to its hydrophobic nature, as such compounds form micelles, which reduces their availability for target binding.Nonetheless, oral administration of sterol-like LXR ligands in animal studies was sufficient to detect them in various tissues and observe their LXR-mediated effects. 23,24,43Even in the absence of systemic effects, orally administered sterol-based LXR ligands were shown to be active in enterocytes, 23,24 where LXR activation reduces cholesterol absorption and induces cholesterol efflux. 9,10The results presented here uncover a potential antiatherogenic benefit of T. prieuriana extracts, as well as other plants containing significant amounts of 1, but also point to possible disadvantages of such extracts originating from activation of LXR in the liver.

■ EXPERIMENTAL SECTION
General Experimental Procedures.IR spectra were recorded on a Bruker Fourier transform/infrared (ATR) spectrophotometer.Exact mass HRESIMS data were recorded using an Agilent 6546 QTOF mass spectrometer (Agilent Technologies, Massy, France) equipped with an ESI source, operating in both positive-and negativeion mode.A BEH Acquity C18 UPLC column (2.1 × 100 mm; i.d.1.8 μm, Waters) was used, with a flow rate of 0.5 mL•min −1 and a linear gradient from 5% B (A: H 2 O + 0.1% formic acid, B: MeCN + 0.1% formic acid) to 100% B over 15 min.Source parameters were set as follows: capillary temperature at 320 °C, source voltage at 3500 V, and sheath gas flow rate at 11 L•min −1 .The divert valve was set to waste for the first 3 min.MS scans were operated in full-scan mode from m/z 100 to 1200 (0.1 s scan time) with a mass resolution of 67,000 at m/z 922.In the positive-ion mode, purine (C 5 H 4 N 4 ) [M + H] + ion (m/z 121.050873) and the hexakis(1H,1H,3Htetrafluoropropoxy)phosphazene (C 18 H 18 F 24 N 3 O 6 P 3 ) [M + H] + ion (m/z 922.009798) were used as internal lock masses.LC-DAD-ELSD analysis of flindissone (1) was conducted on an HPLC (Agilent) equipped with an ELSD (Agilent) using an XSELECT column (4.6 mm × 150 mm, i.d., 5 μm, Waters) with a flow rate of 1 mL•min −1 and a linear gradient from 30% B (A: H 2 O + 0.1% formic acid, B: MeCN + 0.1% formic acid) to 100% B over 15 min.1D NMR spectra were recorded in deuterated chloroform on an AVANCE 300 MHz NMR spectrometer (proton 1 H at 300 MHz and carbon 13 C at 75 MHz).Chemical shifts δ are referenced to residual solvent signals and reported in parts per million (ppm) relative to tetramethylsilane (TMS), and coupling constants J are given in Hz.Column chromatography (CC) was performed using Merck MN silica gel 60 Mesh (0.04−0.063 nm), and thin layer chromatography (TLC) was performed on aluminum silica gel 60 F254 (Merck) precoated plates (0.2 mm layer thickness).Spots were visualized on TLC by a UV lamp (254 and 365 nm) or by heating after spraying with a 20%   Four additional compounds were isolated from the hydroethanolic extracts from the root wood of T. prieuriana by the usual chromatographic techniques.Their structures were established using a combination of 1D and 2D NMR techniques in conjunction with HRESIMS analyses and by comparison with data reported in the literature.Isolated compounds were identified as flindissone (1), deoxyflindissone (2), picraquassin E (3), and prieurianin (4). 4 Moreover, flindissone (1) purity was determined at 95.554% after the integration of its LC-ELSD chromatogram (Figure S3, Supporting Information).
Luciferase Assays.Two types of luciferase assays were performed.One using the full-length human LXR receptors and a luciferase reporter gene driven by an LXR-response element from the ABCA1 gene promoter and a second assay employing hybrid receptors consisting of the LXR-LBD (ligand binding domain) and the yeast transcription factor Gal4-DBD (DNA-binding domain) where the UAS sequence is used as a promoter for luciferase expression.The plasmids used are listed in the Supporting Information.HEK293T cells were transfected with the calcium phosphate method for 6 h.For full-length LXR transfections, 3 μg of receptor, 6 μg of response element, and 3 μg of EGFP were used per 6 million cells, and for Gal4-transfections, 5 μg of Gal4-receptor, 1 μg of response element, and 3 μg of EGFP plasmid were used per 6 million cells.Transfected cells were resuspended in 2.5% charcoal-stripped FBS−DMEM, replated into a 96-well plate to a density of 50,000 cells/well, and treated with compound solutions prepared in the same medium.After an 18 h incubation period, cells were lysed with a commercial lysis buffer from Promega (E3971).Luminescence and fluorescence values were measured on a TECAN Spark Instrument (TECAN Austria, Salzburg, Austria).Luminescence was normalized by fluorescence through division and expressed relative to the vehicle (0.1% DMSO) control ("luciferase activity").EC 50 and E max values were obtained by the nonlinear fitting of the log-transformed concentrations using the variable slope.
Docking.An X-ray crystal structure of the LXRβ-LBD in complex with eCH (PDB 1P8D, resolution 2.80 Å) 34 was utilized for docking with Glide (software version 2021-1, Schrodinger Inc., New York, NY, USA). 36The protein structure was prepared with the Protein Preparation Wizard within the Maestro molecular modeling environment (software version 2021-1, Schrodinger Inc.) 37 using default settings.The preparation included the (i) addition of hydrogen atoms, (ii) assignment of bond orders, (iii) assignment of protonation and metal charge states with Epik, (iv) removal of all chains except for chain A, (v) sampling H 2 O orientations and optimization of the hydrogen bond network, and (vi) restrained minimization using the OPLS4 force field to converge heavy atoms to an RMSD of 0.30 Å.The 3D structures of flindissone and 22R-OHC were prepared with LigPrep within Maestro using the default settings.For docking with Glide, the ligand-binding site was defined within the Receptor Grid Generation wizard to dock ligands similar in size to the cocrystallized ligand.Glide Standard Precision (Glide SP) was used for ligand docking and up to 100 docking poses were set for output.
Cholesterol Efflux Assay.THP-1 monocytes were plated in a 24well plate at a density of 200.000 cells/mL per well and differentiated with 200 nM PMA for 72 h into THP-1 M0 macrophages.Macrophages were first treated with 500 μL of cholesterol solution containing 10 μg of H 2 O-soluble cholesterol (Sigma-Aldrich, C4951) and 0.1 μCi [1,2-3 H(N)]-labeled cholesterol (PerkinElmer, NET139001MC) in 2.5% FBS DMEM medium for 24 h.Cells were washed, and 500 μL of compound solutions were added for another 24 h.Compound solutions were removed, and efflux was stimulated by the addition of 250 μL of 1% plasma in 0.1% BSA-DMEM medium.For each compound treatment, nonstimulated efflux was measured from wells where only 0.1% BSA-DMEM medium was added.After 6 h, the medium was collected and cleared by centrifugation and cells were lysed with 250 μL of 0.1N NaOH for 10 min.Media and lysates were mixed with 3.5 mL of scintillation counting liquid (PerkinElmer, 6013329).The efflux values were calculated as follows:% Efflux (1% plasma or blank) = [ 3 H] (medium)/[ 3 H] (medium) + [ 3 H] (cells) * 100.Plasma-mediated % Efflux = % Efflux (1% plasma) − % Efflux (blank).Plasma-mediated % Efflux for compound treatments was normalized to the DMSO by subtraction.
Compound Treatments.For RNA and protein extractions, 800.000THP-1 macrophage cells plated in six-well plates (after 72 h differentiation) were first treated with 2 mL of cholesterol solution containing 40 μg of H 2 O-soluble cholesterol in 2.5% FBS−DMEM for 24 h before treatment with compounds, to keep consistency with the Cholesterol efflux assay.For the HepG2 cell line, 600.000 cells were plated in 4 mL per well and incubated with compounds from the following day in a 2 mL solution for 72 h, with fresh compound solution added every 24 h.All compounds were diluted in 0.1% BSA-DMEM medium to an indicated final concentration.The final concentration of the solvent (DMSO) was kept constant at 0.1% for all compounds and concentrations.
mRNA Quantification.RNA was extracted with the innuPREP RNA Mini Kit 2.0 kit (IST Innuscreen, 845-KS-2040250) according to the manufacturerś protocol.One μg of RNA was used for reversetranscriptase reaction (Applied Biosystems, 4368814) and the cDNA template was diluted to a final volume of 50 μL.Two μL of the template was used for the qPCR reaction according to the manufacturerś protocol (Promega, A6002).All primers except for the human GAPDH (Qiagen, 249900) were designed with Primer-BLAST and were tested for efficiency and specificity before use.The sequences can be found in the Supporting Information.The "relative gene expression" was calculated using the ddCt method and expressed in comparison to the 0.1% DMSO control.
Oil Red O Staining.HepG2 cells were plated and treated with compounds as described in Compound treatments.Cells were fixed with 10% formaldehyde for 10 min at room temperature, rinsed with PBS, permeated with 60% isopropanol, and stained with 60% Oil Red O solution for 1 min at 37 °C.After 5 washes with PBS and drying, cells were photographed, and the dye was eluted with 100% isopropanol.OD values were measured at 492 nm, and the results were expressed relative to the DMSO sample.
Statistical Analysis.All of the experiments were performed independently at least three times.Luciferase experiments were further performed with 4 technical replicates per experiment.Data analysis was performed in MS Excel, and graphs and statistical analysis were performed in GraphPad Prism v8 (GraphPad, La Jolla, CA, USA).For all graphs: Bars and whiskers display mean ± SD; unless otherwise indicated in the legend.Bullet points are individual values (N).Statistical test: One-way ANOVA with Dunnett's post hoc test.Significance indicated was in comparison to the DMSO (0.1%) control.
■ ASSOCIATED CONTENT * sı Supporting Information

Figure 2 .
Figure 2. Depiction of the likely binding mode of (A) 22R-hydroxycholesterol (22R-OHC; green carbon atoms) and (B) flindissone (1; cyan carbon atoms) for the LXRβ ligand binding domain, derived by docking.24(S),25-Epoxycholesterol (eCH), the ligand present in the cocrystal structure used for docking (PDB 1P8B), 34 is depicted with gray carbon atoms.Magenta dashed lines indicate hydrogen bond interactions; the numbers indicate distances in Å.Amino acid residues forming hydrogen bonds with the ligands via H 2 O molecules are marked by the thick tube representation.